Method for obtaining fetal cells and fetal cellular components

ABSTRACT

Methods are disclosed for non-invasively obtaining fetal cells from a pregnant female. The methods include placing an absorbent medium in an interlabial or intravaginal space or adjacent to the perineum at the vaginal opening of the pregnant female, and collecting vaginal fluid comprising cells in the absorbent medium while the absorbent medium is interlabial or intravaginal space or adjacent to the perineum at the vaginal opening. The absorbent medium is removed and cells are isolated from the absorbent medium to obtain the fetal cells. The fetal cells can be, for example, somatic cells, embroyic stem cells, fetal stem cells or trophoblast cells.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/663,456, filed Jun. 22, 2012, which is herein incorporated byreference in its entirety.

FIELD

This relates to the field of cell purification, specifically to methodsfor isolating fetal cells from a pregnant woman using non-invasivemethods.

BACKGROUND

Amniocentesis is a medical procedure used in prenatal diagnosis ofchromosomal abnormalities and fetal infections, in which a small amountof amniotic fluid, which contains fetal tissues, is obtained from theamnion or amniotic sac, and the fetal DNA is examined for geneticabnormalities. This process also can be used for prenatal sexdiscernment.

Amniocentesis is generally performed between the 15th and 20th week ofpregnancy; performing this test earlier may result in fetal injury. Theterm “early amniocentesis” is sometimes used to describe use of theprocess between weeks 11 and 13. However, it is not possible to useamniocentisis to obtain DNA or fetal cells from a fetus of less than 11weeks of gestation. In addition, amniocentesis is invasive.

Amniotic fluid is a source of multipotent mesenchymal, hematopoietic,neural, epithelial, and endothelial stem cells. However, collectingthese cells can result in complications. Artificial heart valves,working tracheas, as well as muscle, fat, bone, heart, neural and livercells have been produced from stem cells isolated from amniotic fluid.Tissues obtained from amniotic cell lines show promise for patientssuffering from congenital diseases/malformations of the heart, liver,lungs, kidneys, and cerebral tissue.

However, complications of amniocentesis include preterm labor anddelivery, respiratory distress, postural deformities, fetal trauma andalloimmunisation of the mother (rhesus disease). Studies from the 1970soriginally estimated the risk of amniocentesis-related miscarriage ataround 1 in 200 (0.5%) although other more recent studies estimated theprocedure-related pregnancy loss at 0.6-0.86%. Thus, a need remains forother methods that can be used to collect fetal stem cells and that canbe used for fetal diagnosis.

SUMMARY

Disclosed are completely new uses of external collection devices,including absorbent interlabial pads, sanitary napkins and tampons.Specifically, it is disclosed herein that these devices can be used forthe collection of fetal cells, such as, but not limited to, somaticcells, embryonic stem cells, fetal stem cells or trophoblast cells.Surprisingly, it was determined that fetal cells remain viable in thesedevices, and can be isolated and propagated following collection. Inaddition, these devices can be used for the collection of the componentsof fetal cells, such as DNA, RNA, proteins and lipids.

In some embodiments, methods are provided for obtaining fetal cellsand/or fetal cell components from a pregnant female. The methods includeplacing an absorbent medium in an interlabial or intravaginal space oradjacent to the perineum at the vaginal opening of the pregnant female,and collecting vaginal fluid comprising fetal cells and/or fetalcellular components in the absorbent medium while the absorbent mediumis in the interlabial or intravaginal space or adjacent to the perineumat the vaginal opening. The absorbent medium is removed and cells areisolated from the absorbent medium to obtain the fetal cells. The fetalcells can be, for example, somatic cells, embryonic stem cells, fetalstem cells or trophoblast cells.

In additional embodiments, methods are provided for fetal diagnosis. Themethods include placing an absorbent medium in an interlabial orintravaginal space or adjacent to a perineum at a vaginal opening of apregnant female, and collecting vaginal fluid in the absorbent medium.The absorbent medium is removed from the pregnant female, and cells inthe absorbent medium are subjected to a fetal diagnostic test. In someembodiments, the method includes isolating at least one fetal cell fromthe absorbent medium. The genetic material, or genetic material isolatedfrom the at least one fetal cell can be analyzed to determine thepresence or absence of a genomic or epigenetic characteristic associatedwith a biological outcome. In some embodiments, methods are provided fordetermining the sex of a fetus.

The foregoing and other features and advantages of this disclosure willbecome more apparent from the following detailed description of severalembodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are a set of flow charts illustrating an embodiment ofprocessing of absorbent medium for the collection of fetal cells.

FIG. 2 is an analysis of genetic material showing the presence of maleDNA in samples collected from pregnant females known to be carryingboys. A single-center study was performed, focused on OB/GYN Centerswith access to pregnant patients with an identified male fetus. Thedesign of this study involved home-based sample collection, and includedblinded testing from study samples. Diagnosis was compared to ultrasoundoutcome. Subjects were screened based on ultrasound results. Womenenrolled in the study were required to collect two (2) vaginal samples,within 14 days of the ultrasound results. There were no abnormalclinical follow-ups after completion of this study.

FIG. 3 is an additional analysis of genetic material showing thepresence of male DNA in samples collected from pregnant females known tobe carrying boys.

FIG. 4 is a digital image of a cytology evaluation. Upon harvest as apellet, ½ of each PADKIT® sample was subjected to digital cytometryusing standard HOLOGICS® Thin-prep technology and Pap staining performedat a CLIA-certified Cytometry lab. These Thin-prep slides were thenanalyzed by APERIO® digital Cytometry. Both standard light and digitalcytometry confirmed that all samples processed for this study showedordinary cell morphology, which were indistinguishable relative toroutine PAP stained cervical scrapes.

FIG. 5 is a digital image of a second cytology evaluation.

FIG. 6 is a schematic view of the perineum and thighs, which illustratesthe external female genitalia.

FIG. 7 is a cross-sectional sagittal view taken along line 2-2 of FIG.1, but showing a pad positioned between the labia.

FIG. 8 is a schematic view of one embodiment of the interlabial pad.

FIG. 9 is a cross sectional fragmentary view of another embodiment ofthe interlabial pad.

FIGS. 10-14 are views similar to FIG. 4, but showing differentembodiments of the pad which have a substantially quadrilateral shape ormajor portion.

FIGS. 15 and 16 are cross sectional views of the major portion of thepad, showing the major portion to be either arcuate (FIG. 15) or tapered(FIG. 16).

FIG. 17 is a cross sectional view of an interlabial absorbent pad thatdoes not have a major portion and a minor portion, but which has theside surfaces of the pad sloping toward a leading edge of the pad.

FIGS. 18-20 are cross-sectional fragmentary views showing pads, whichhave major portions that are polygonal in shape.

FIG. 21 is a cross sectional view of an elongated interlabial pad with amajor portion and a minor portion, both of which taper symmetrically ina longitudinal direction.

FIG. 22 is a view similar to FIG. 16, but showing the major and minorportions of the pad tapering longitudinally in different directions.

FIG. 23 is a perspective view of an elongated interlabial absorbent padthat has a fixed diameter along the length of the pad.

FIGS. 24-26 are side views of interlabial absorbent pads similar to thepad shown in FIG. 23, but with one or two sloping end edges.

FIG. 27 is a cross sectional view of an interlabial absorbent padwherein the posterior portion of the pad is formed with a longitudinalgroove.

FIG. 28 is a cross sectional view of the interlabial absorbent pad ofFIG. 22 disposed between the labia in the interlabial space.

FIG. 29 is a cross sectional view of a unitary, one-piece yet bipartiteinterlabial absorbent pad in which each portion of the pad has a crosssection of a portion of a circle, each circle having different radii ofcurvature.

FIG. 30 is a cross sectional view of a bipartite pad in which eachportion of the pad has a cross section of a partial ellipse. The padsmay be either symmetric or asymmetric. In the symmetric embodiment, themajor and minor portions may have the shape of partial spheres orellipsoids.

FIG. 31 is a cross sectional view of an additional embodiment of aone-piece interlabial pad with an elliptical cross section, and no minorand major portions.

FIG. 32 is an end perspective view of an elongated pad with a minor anda major portion that extends along its length, and a groove in the minorportion from which drugs or other agents can be released by compressionof the pad in use.

FIG. 33 is an end view of the pad of FIG. 27.

FIG. 34 is a perspective view of an elongated folded pad.

FIG. 35 is an end view of the pad of claim 29.

FIG. 36 is a perspective view of an elongated pad.

FIG. 37A is an MRI of an external feminine hygiene pad in place againstthe external female genitalia.

FIG. 37B is an MRI of an example of a pad in accordance with the presentdisclosure, in which the pad is retained between the labia, external tothe hymenal ring.

DETAILED DESCRIPTION

Methods are disclosed for non-invasively obtaining fetal cells from apregnant female. The methods include placing an absorbent medium in aninterlabial or intravaginal space or adjacent to the perineum at thevaginal opening of the pregnant female, and collecting vaginal fluidcomprising cells in the absorbent medium while the absorbent medium isinterlabial or intravaginal space or adjacent to the perineum at thevaginal opening. The absorbent medium is removed and cells are isolatedfrom the absorbent medium to obtain the fetal cells. The fetal cells canbe, for example, somatic cells, embryonic stem cells, fetal stem cellsor trophoblast cells.

I. TERMS

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Absorbent: A material with sufficient absorbency to absorb and retainexudates discharged from a subject, such as fluids and/or cells.Absorbency is dependent partially on the physical volume of the device.In a specific non-limiting example, a material is absorbent if itabsorbs at least 3 ml of 0.9% saline, however an absorbent material mayhave a capacity of 20 grams or more.

Agent: A substance capable of producing a physical, chemical orbiological effect. Examples of agents include drugs (therapeutic agents)and diagnostic reagents (diagnostic agents). Examples of drugs includeantimicrobial agents (such as the anti-fungal agent miconazole,anti-viral acyclovir, or anti-biotic metronidazole). Examples ofdiagnostic agents include monoclonal antibodies (such as monoclonalantibodies that recognize pathologic agents, such as viruses, chemicalreagents in which a reaction occurs in the presence of a pathogen ofinterest, such as a color change), or agents that can be used todiagnose.

Amniotic Sac: The membranes that a fetus develops in amniotes. The innermembrane is the amnion, and the outer membrane is the chorion. On theouter side, the amniotic sac is connected to the allantois and yolk sac,and, through the umbilical cord, to the placenta. An “intact” amnioticsac includes unbroken membranes containing the amniotic fluid. A“ruptured” amniotic sac is one with a broken membrane.

Amplification: To increase the number of copies of a nucleic acidmolecule. The resulting amplification products are called “amplicons.”Amplification of a nucleic acid molecule (such as a DNA or RNA molecule)refers to use of a technique that increases the number of copies of anucleic acid molecule in a sample. An example of amplification is thepolymerase chain reaction (PCR), in which a sample is contacted with apair of oligonucleotide primers under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, re-annealed, extended, and dissociated to amplify the numberof copies of the nucleic acid. This cycle can be repeated. The productof amplification can be characterized by such techniques aselectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region which specifically recognizesand binds an epitope of an antigen. Antibodies are composed of a heavyand a light chain, each of which has a variable region, termed thevariable heavy (V_(H)) region and the variable light (V_(L)) region.Together, the V_(H) region and the V_(L) region are responsible forbinding the antigen recognized by the antibody.

This includes intact immunoglobulins and the variants and portions ofthem well known in the art, such as Fab′ fragments, F(ab)′₂ fragments,single chain Fv proteins (“scFv”), and disulfide stabilized Fv proteins(“dsFv”). A scFv protein is a fusion protein in which a light chainvariable region of an immunoglobulin and a heavy chain variable regionof an immunoglobulin are bound by a linker, while in dsFvs, the chainshave been mutated to introduce a disulfide bond to stabilize theassociation of the chains. The term also includes genetically engineeredforms such as chimeric antibodies (for example, humanized murineantibodies), heteroconjugate antibodies (such as, bi-specificantibodies). See also, Pierce Catalog and Handbook, 1994-1995 (PierceChemical Co., Rockford, Ill.); Kuby, J., Immunology, 3^(rd) Ed., W.H.Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (k). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs.” The extent of theframework region and CDRs have been defined (see, Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991, which is hereby incorporated byreference). The Kabat database is now maintained online The sequences ofthe framework regions of different light or heavy chains are relativelyconserved within a species. The framework region of an antibody, that isthe combined framework regions of the constituent light and heavychains, serves to position and align the CDRs in three-dimensionalspace. The CDRs are primarily responsible for binding to an epitope ofan antigen.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies. A “chimeric antibody” has framework residues from onespecies, such as human, and CDRs (which generally confer antigenbinding) from another species, such as a murine antibody. A “human”antibody (also called a “fully human” antibody) is an antibody thatincludes human framework regions and all of the CDRs from a humanimmunoglobulin. In one example, the framework and the CDRs are from thesame originating human heavy and/or light chain amino acid sequence.However, frameworks from one human antibody can be engineered to includeCDRs from a different human antibody. A “humanized” immunoglobulin is animmunoglobulin including a human framework region and one or more CDRsfrom a non-human (for example a mouse, rat, or synthetic)immunoglobulin. The non-human immunoglobulin providing the CDRs istermed a “donor,” and the human immunoglobulin providing the frameworkis termed an “acceptor.” In one embodiment, all the CDRs are from thedonor immunoglobulin in a humanized immunoglobulin. Constant regionsneed not be present, but if they are, they must be substantiallyidentical to human immunoglobulin constant regions, i.e., at least about85-90%, such as about 95% or more identical. Hence, all parts of ahumanized immunoglobulin, except possibly the CDRs, are substantiallyidentical to corresponding parts of natural human immunoglobulinsequences. A “humanized antibody” is an antibody comprising a humanizedlight chain and a humanized heavy chain immunoglobulin. A humanizedantibody binds to the same antigen as the donor antibody that providesthe CDRs. The acceptor framework of a humanized immunoglobulin orantibody may have a limited number of substitutions by amino acids takenfrom the donor framework. Humanized or other monoclonal antibodies canhave additional conservative amino acid substitutions which havesubstantially no effect on antigen binding or other immunoglobulinfunctions. Humanized immunoglobulins can be constructed by means ofgenetic engineering (see for example, U.S. Pat. No. 5,585,089).

Aneuploidy: An abnormal number of chromosomes. Monosomy refers to thepresence of only one chromosome, wherein two copies is normal. Monosomyof the X chromosome (45,X) causes Turner's syndrome. Trisomy refers tothe presence of three copies (instead of the normal two) of specificchromosomes. Trisomy 21 causes Down's syndrome. Tripsome 10 and Trisomy31, known as Edwards and Patau Syndrome, respectively, are two autosomalabnormalities. Trisomy X has also been observed in humans (47, XXX).

Germline aneuploidy can be detected through karyotyping, a process inwhich a sample of cells is fixed and stained to create the typical lightand dark chromosomal banding pattern and a picture of the chromosomes isanalyzed. Other techniques include Fluorescence In Situ Hybridization(FISH), Quantitative Polymerase Chain Reaction (PCR) of Short TandemRepeats, Quantitative Fluorescence PCR (QF-PCR), Quantitative Real-timePCR (RT-PCR) dosage analysis, Quantitative Mass Spectrometry of SingleNucleotide Polymorphisms, and Comparative Genomic Hybridization (CGH).

Biodegradable material: A material having greater than or equal to about70% biodegradation (percentage of theoretical carbon dioxide evolution)after 28 days when measured by a suitable test such as the Sturm test(Method 301B, Organization of Economic Cooperation and Development).

Cellular Components: The biological molecules, such as DNA, RNA, lipids,protein and phospholipids that are contained in a cell. “Fetal cellularcomponents” are biological molecule, such as DNA, RNA, lipids andproteins isolated from fetal cells. Similarly, “maternal cellularcomponents” are biological molecule, such as DNA, RNA, lipids andproteins isolated from cells of a mother of a fetus or baby, such as apregnant female. “Genetic material” includes both DNA and RNA.

Diagnostic test: Any procedure performed on a biological sample, whereinthe procedure can be used to evaluate or monitor a disease or adisorder, or can be used to determine the genotype. Diagnostic testsinclude tests that analyze the genomic and/or epigenetic characteristicsof a fetus by analyzing fetal cells and/or cellular components, such asDNA or proteins. A diagnostic test can be performed in a laboratory, amedical office or in the home environment. A diagnostic test can also beused to determine fetal sex.

DNA methylation: The post synthetic addition of methyl groups tospecific sites on DNA molecules; the reaction is catalyzed by enzymescalled DNA methyltransferases that are specific for nucleotide andposition of methylation. In eukaryotes, methylation is involved in geneexpression, and plays a role in a variety of epigenetic mechanisms,including development, X chromosome inactivation, genomic imprinting,mutability of DNA, and uncontrolled cell growth in cancer. The term “Xchromosome inactivation” refers to the inactivation of one of each pairof X chromosomes to form the Barr body in female mammalian somaticcells. Thus tissues whose original zygote carried heterozygous X bornegenes should have individual cells expressing one or other but not bothof the X encoded gene products. The inactivation is thought to occurearly in development and leads to mosaicism of expression of such genesin the body.

Embryonic Stem (ES) Cells: Pluripotent stem cells derived from the innercell mass of the blastocyst that proliferate in vitro. Human embryosreach the blastocyst stage 4-5 days post fertilization, at which timethey consist of 50-150 cells. ES cells. Because of their plasticity andpotentially unlimited capacity for self-renewal, ES cell therapies havebeen proposed for regenerative medicine and tissue replacement afterinjury or disease. In some embodiments, ES cells can be produced fromculturing the inner cells mass of a blastocyst and culturing these cellson fibroblasts in the presence of mitomycin-C in serum containingmedium. These cells can form embryoid bodies.

Epigenetic: A heritable change in gene expression or cellular phenotypecaused by mechanisms other than changes in a DNA sequence. Examples ofsuch changes are DNA methylation and histone modification. Method ofdetecting such modifications are disclosed, for example, in U.S. Pat.Nos. 5,625,105; 6,300,071; 6,569,684; 7,035,739; 7,037,650; and7,144,701.

Fetal cells: Cells from a fetus. In several embodiments, fetal cells arepresent in vaginal fluid collected from a pregnant female. The fetalcells present in the vaginal fluid can be isolated from maternal cellsalso present in the vaginal fluid.

Fetus: Unborn offspring of a female mammal more than 8 weeks afterconception.

Genomic or epigenetic characteristic associated with a biologicaloutcome: A genetic characteristic or epigenetic characteristic thatresults in, or is correlated with, a phenotype of a subject. The geneticcharacteristic can be the presence or absence of all or a portion of achromosome (e.g., an aneuploidy) or the presence or absence of a geneticmutation, that is associated with a particular biologicalcharacteristic, such as sex or disease state. Similarly, an epigeneticcharacteristic can be a heritable change in gene expression or cellularphenotype, caused by a mechanism other than changes in a DNA sequence,that is associated with a particular biological characteristic, such assex or disease state.

Intralabial Pad: Absorbent pad designed to be placed longitudinallybetween the vaginal lips or labia, and are particularly useful to absorbvaginal discharges.

Invasive Collection of Fetal Cells: A procedure that involvespenetration of the cervix, biopsy, or penetration the skin (such as witha needle), of a pregnant female for the collection of fetal cells.Amniocentesis, chorionic villus sampling, and transcervical cellcollection are examples of invasive methods of fetal cell collection. A“non-invasive” method for collecting cells does not involve penetrationof the cervix, biopsy, or penetration of the skin of a subject for cellcollection. The collection of cells by placing an absorbent medium in aninterlabial or intravaginal space or adjacent to the perineum at thevaginal opening of the pregnant female, and isolating cells from theabsorbent medium is a non-invasive method of collection fetal cells.

Isolated Cells or Purified Cells: As used herein, the term “isolatedcells,” “purified cells,” “isolated cell population,” “purified cellpopulation” refers to a preparation of one or more cells, such as fetalcells, that has been manipulated to provide a preparation of cells thatis substantially free of additional components, such as maternal cells.In some embodiments, the fetal cells are at least about 70%, by weightor number, free from other components that are present when the cell isproduced, such as other types of cells (e.g., maternal cells). Invarious embodiments, the cell is at least about 75%, or at least about85%, or at least about 90%, or at least about 95%, or at least about99%, by weight or number, pure, from maternal cells. A purified cellpreparation can be obtained, for example, by purification (e.g.,extraction) using fluorescence-activated cell-sorting or magnetic beadaffinity purification, or other techniques known to the skilled artisan.Purity can be assayed by any appropriate method, such asfluorescence-activated cell-sorting (FACS) or by visual examination.

Larger and smaller portions: The major portion of the pad is a largerportion, and a minor portion is a smaller portion. Large and small canbe defined, for example, in terms of cross-sectional area, volume, ortransverse dimension. In some embodiments, the pad is inserted betweenthe labia with the minor portion as the leading edge inserted, in whichexample the minor portion would also be considered an anterior edge andthe major portion would be a posterior portion.

Medicinal Agent: A therapeutic agent for treatment of the interlabialspace, perivaginal region, vagina, and/or for delivery or for cellpreservation. Specific, non-limiting examples of a medicinal agent areanesthetics, lubricants or preservatives.

Totipotent, Pluripotent, Multipotent Stem Cells: As used herein, theterm “totipotent” or “totipotency” refers to a cell's ability to divideand ultimately produce an entire organism including extra-embryonictissues in vivo. In one aspect, the term “totipotent” refers to theability of the cell to progress through a series of divisions into ablastocyst in vitro. The blastocyst comprises an inner cell mass (ICM)and a trophoblast. The cells found in the ICM give rise to pluripotentstem cells (PSCs, see below) that possess the ability to proliferateindefinitely, or if properly induced, differentiate in all cell typescontributing to an organism. Trophoblast cells generate extra-embryonictissues, including placenta and amnion.

Totipotent Stem cells (TSCs) are the source of PSCs. As used herein, theterm “pluripotent” refers to a cell's potential to differentiate intocells of the three germ layers: endoderm (e.g., interior stomach lining,gastrointestinal tract, the lungs), mesoderm (e.g., muscle, bone, blood,urogenital), or ectoderm (e.g., epidermal tissues and nervous system).Pluripotent stem cells can give rise to any fetal or adult cell typeincluding germ cells. However, PSCs alone cannot develop into a fetal oradult animal when transplanted in utero because they lack the potentialto contribute to extra-embryonic tissue (e.g., placenta in vivo ortrophoblast in vitro).

PSCs are the source of multipotent stem cells (MPSCs) throughspontaneous differentiation or as a result of exposure todifferentiation induction conditions in vitro. The term “multipotent”refers to a cell's potential to differentiate and give rise to a limitednumber of related, different cell types. These cells are characterizedby their multi-lineage potential and the ability for self-renewal. Invivo, the pool of MPSCs replenishes the population of maturefunctionally active cells in the body. Among the exemplary MPSC typesare hematopoietic, mesenchymal, or neuronal stem cells. “Committedprogenitors” give rise to a fully differentiated cell of a specific celllineage. Exemplary lineages include pancreatic cells, epithelial cells,cardiac cells, endothelial cells, liver cells, endocrine cells, and thelike.

Trophoblast: The outermost layer of cells of the embryo of placentalmammals that attaches the fertilized ovum to the uterine wall.Trophoblasts play an important role in embryo implantation andinteraction with the decidualised maternal uterus. The core of placentalvilli contain mesenchymal cells and placental blood vessels that aredirectly connected to the fetal circulation via the umbilical cord. Thiscore is surrounded by two layers of trophoblast; a single layer ofmononuclear cytotrophoblast that fuse together to form the overlyingmultinucleated syncytiotrophoblast layer that covers the entire surfaceof the placenta. It is this syncytiotrophoblast that is in directcontact with the maternal blood that reaches the placental surface, andthus facilitates the exchange of nutrients, wastes and gases between thematernal and fetal systems.

Vaginal fluid: Aqueous solution secreted or discharged from the vagina.Vaginal fluid can include cells. Vaginal fluid from a pregnant femalecan include maternal cells and cellular components, as well as fetalcells and fetal cellular components. For example, the fetal cells can besomatic cells, embryonic stem cells, fetal stem cells or trophoblasts.In some embodiments, the fetal cells can be totipotent, multipotent orpluripotent stem cells.

Vaginal orifice: The opening of the vagina at the perineum.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of this disclosure, suitable methods and materials are describedbelow. The term “comprises” means “includes.” In case of conflict, thepresent specification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

II. COLLECTING FETAL CELLS AND/OR CELLULAR COMPONENTS

Methods are provided herein for the collection of fetal cells and/orcomponents of fetal cells. These methods include placing an absorbentmedium in an interlabial or intravaginal space or adjacent to theperineum at the vaginal opening of a pregnant female and collectingvaginal fluid comprising cells and/or cellular components in theabsorbent medium while the absorbent medium is in the interlabial orintravaginal space or adjacent to the perineum at the vaginal opening.The fetal cells and/or cellular components can be collected at any timeduring gestation, including during the first, second and thirdtrimester. In some embodiments, the fetal cells and/or cellularcomponents are collected in the absence of rupture of the amniotic sac.In other embodiments, the fetal cells and/or cellular components arecollected without collecting maternal blood.

These methods allow fetal cells to be obtained without invasion of thecervix. Thus, transcervical sampling is not utilized. In someembodiments, the pregnant female has an intact amniotic sac. In otherembodiments, the pregnant female has a rupture in the amniotic sac.

The fetal cells can be any cells of interest, including somatic cells,embryonic stem cells, fetal stem cells or trophoblasts. The fetal cellscan be totipotent, multipotent or pluripotent stem cells. The cellularcomponents can be any of the biological components of a cell, including,but not limited to DNA, RNA, proteins and lipids.

The absorbent medium can be used in the form of an interlabial pad,sanitary napkin or panty-liner. The retention of an interlabial pad inthe interlabial space, or the use of a sanitary napkin or panty-liner,permits sustained contact between the pad and the vaginal orifice, forcollection of fluids and fetal cells. Similarly a tampon can be retainedin the vaginal canal for a sufficient time for the collection of fluidsand fetal cells. In some embodiments, the pad is retained for a periodof time that is sufficient for the collection of fetal cells, such asabout 2 hours to about 10 hours, such as about 2 to about 8 hours, suchas about 4 to 6 hours. In some specific non-limiting examples, the pad(or tampon) is retained such as about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, or about 12hours. The absorbent medium can have an inner core and an outercovering, wherein the outer covering has a visible matrix of pores ofsufficient size to allow fetal cells to enter the pores. In someembodiments, the pores are at least 10 μm in diameter, such as about 10μm to about 20 μm in diameter. In some embodiments, this is configuredfor intravaginal or interlabial placement.

In some embodiments, the methods utilize an interlabial pad. Variousforms of interlabial pads, as well as methods of producing them, aredescribed in U.S. Pat. No. 3,983,873; U.S. Pat. No. 4,095,542; U.S. Pat.No. 4,142,476; U.S. Pat. No. 4,995,150; U.S. Pat. No. 5,575,047; U.S.Pat. No. 5,727,481; U.S. Pat. No. 6,007,498; U.S. Pat. No. 6,183,455;U.S. Pat. No. 6,811,549; which are all incorporated herein by reference.These pads are designed to be placed longitudinally between the vaginallips or labia, and are particularly useful to absorb light discharges ofmenstrual fluids, mid-cycle spotting or discharges, slight loss of urinecaused by physical stress, or leakages following intercourse. In someembodiments, a biodegradable interlabial pad is utilized. Thebiodegradable pad is capable of being decomposed by natural biologicalprocesses.

Another example of an interlabial pad suitable for use with the presentmethod is the absorbent interlabial device disclosed in U.S. Pat. No.5,968,026, which issued to the Procter & Gamble Company, and which isincorporated by reference herein. A commercially available example ofthis pad is the Envive Miniform.

However, the invention is not limited to these specific particularlydisclosed embodiments, which are only given by way of illustration. Anyconfiguration of the pad is possible, which allows it to be capable ofbeing substantially retained in the interlabial space by engagement withthe labial folds, but can be simply and easily removed by manuallyremoving it.

The interlabial pad is positioned such that the pad is retained betweenthe labia external to the subject's vagina. The anterior portion of thepad is designed for insertion of the pad between the subject's labia inthe anatomic interlabial space adjacent to the vaginal orifice, and theposterior portion is retained between the labia without the need foradhesive or other attachment devices, as in FIG. 37B. Alternatively, aportion of the pad can project into the vagina, for example to improveretention and enhance cell collection.

The interlabial pad can be any of a variety of shapes, and particularlyshapes which taper toward an anterior or leading edge of the pad. Theanterior edge is often sufficiently wide to be retained outside thevaginal orifice, but can be sufficiently narrow to extend at leastpartially within the vagina (for example no more than 1 inch into thevagina, and in some examples less than ½ inch). When the pad issubstantially or completely retained external to the vagina, theposterior edge impinges against the surrounding labia to retain the padin place. The pad can be symmetric or asymmetric, rounded or elongated,tapering or non-tapering, folded or not folded. However, particularembodiments taper from a relatively larger posterior portion to arelatively smaller anterior portion. The enlarged posterior portion isoften large enough to at least slightly deform the surrounding labia toimprove frictional engagement between the labia and the pad. Therelatively small anterior portion may in some examples be closer to thewidth of the vaginal orifice, and is more comfortably retained in thenarrow interlabial space adjacent the vaginal orifice. The pads with abipartite structure (with a major and minor portion) further enhance thecomfort and retention of the pad. The approach disclosed herein isnon-invasive route (for example, avoiding the risks of transcervicalcollection).

An embodiment is shown in FIGS. 6-8. FIG. 6 illustrates the urogenitalanatomy of a female. Interlabial space 32 is approximately bounded bythe labia majora 34. Anatomical structures found within the interlabialspace include the labia minora 36, vaginal orifice 38, urethral orifice40, and clitoris 42. The perineum is a term that often refers to thepelvic outlet that gives passage to the urogenital ducts and anus, butit is used herein n a more restricted sense to refer to the area 44which lies between interlabial space 32 and the anus 46. A perineal padabuts against at least a portion of the perineum.

FIG. 7 is a sagittal section of female urogenital anatomy, andillustrates that in this embodiment of the invention, interlabial pad 30is positioned in interlabial space 32 approximately adjacent labiamajora 34, vaginal orifice 38, and urethral orifice 40.

In the embodiments disclosed in FIGS. 7-8, interlabial pad 30 is anelongated absorbent member, for example made of cotton, and has abipartite profile with a major portion 50 and a minor portion 52. In theillustrated example, the major and minor portions each have a crosssection that is a portion of a circle, where the portion of the circleof the major portion 50 has a greater diameter than the portion of thecircle of the minor portion 52. The curvature of the minor portion isgreater than the curvature of the major portion. The overall shape ofpad 30 therefore includes a rounded major portion and a rounded minorportion, in which the transverse diameter or width W1 (FIG. 8) of themajor portion is greater than the transverse diameter or width W2 of theminor portion, so that the width of pad 30 tapers in the direction ofminor portion 52.

The width of major portion 50 is sufficient to fit comfortably and beretained without adhesives within the interlabial space. Minor portion52 has a reduced width (and increased taper) to minimize pressure anddiscomfort in the area of vaginal and urethral orifices 32. The minimumwidth of minor portion 52 is, in some embodiments, substantially thesame or slightly less than the maximum diameter of vaginal orifice 38.The outer profile of both the major and minor portions may be arcuate tohelp conform to surrounding body tissues. The cross-sectional area ofminor portion 52 in some embodiments is less than 50% of thecross-sectional area of pad 30, and has a cross-sectional area that is,for example, 10 to 49% of the total cross-sectional area of pad 30.

The reduced width of minor portion 52 makes interlabial pad 30 easy toinsert and use. Labia majora 34 and labia minora 36 are spread aparteither by moving them apart, or by introducing the reduced width minorportion 52 as a leading edge of the pad between them, and advancing thepad toward vaginal orifice 38. As pad 30 is inserted into interlabialspace 32, the leading minor portion 52 gradually moves labia majora andlabia minora apart, to facilitate acceptance of major portion 50. Onceminor portion 52 is in place against vaginal orifice and urethralorifice 32, major portion 50 provides an enlarged retention member thatfrictionally engages surrounding portions of labia majora 34 to retaininterlabial pad 30 in position.

The pad is easily inserted between the labia majora and is easilyretained in the interlabial space without the need for auxiliaryretaining means. Thus, a light pressure on the major portion 50 willcause the smaller minor portion 52 to open the labia majora slightly andallow pad 30 to take its proper position in the interlabial space. Theradii of the respective portions are such that the interlabial space 36is substantially or completely occupied by the pad. The elongated padextends along the interlabial space, such that the length of the padhelps frictionally engage the pad and enable it to resist dislodgementso that fetal cells can be collected.

Some other examples of alternative embodiments of the pad with atapering portion are shown in FIGS. 9-34. Many of these embodiments areshown in cross-section as relatively flat, although they can beelongated (as indicated by the fragmentary depiction in each Figure).

In the embodiment shown in FIG. 9, a one piece absorptive pad 58 has a“tear-drop” or ovoid cross sectional shape which tapers progressively toa leading anterior edge portion 60 of limited transverse dimension froma posterior portion 62 of relatively large transverse dimension. The pad58 may be elongated transverse to the illustrated cross-section, or itmay not be elongated (such that the length of the pad transverse to thecross section is less than the anterior-posterior dimension A-P of thecross-section). In elongated embodiments, the pad may be of uniformcross section along the length thereof, or may be tapered from one endto the other end thereof, and in particular embodiments, is tapered inits anterior-posterior dimension AP. The user may readily and quicklyinsert the pad 58 into the interlabial space by introducing leadinganterior portion 60 into the interlabial space. The pad is firmly selfretained in the space and exhibits substantial absorptive capacity forvaginal fluid and fetal cells, and resists accidental dislodgement fromthe interlabial space.

Other embodiments of the pad are shown which have posterior majorportions of a polygonal (for example quadrilateral) shape, such asrectangular or square. Thus, as shown in FIG. 10, pad 62 includes aposterior portion 64 having flat bottom and side surfaces; and theanterior minor portion 66 has surfaces 68 which incline toward oneanother toward a leading edge 70. Anterior portion 66 therefore forms awedge that parts the labia as it is introduced between them.

FIG. 11 shows a pad 72 that includes a posterior portion 74 ofsubstantially square cross section; and a fingerlike anterior portion 76of limited transverse dimension, which is much narrower than thecorresponding transverse dimension of posterior portion 74. The juncture78 of portions 74, 76 forms an essentially flat shoulder that extendstransverse to the anterior-posterior dimension AP. In the disclosedembodiment, the anterior-posterior dimension of anterior portion 76 issubstantially the same as the anterior-posterior dimension of posteriorportion 74. The slender projecting finger of this pad can be configuredto project through the vaginal orifice and into the vagina when theinterlabial pad is in place. As discussed in detail below, theprojecting finger can carry an agent designed to increase cellviability. In some embodiments, the projecting figure includes anon-toxic core material, such as, but not limited to, rayon.

FIG. 12 shows a pad 80 that is similar to that of FIG. 11, except thatthe sides of anterior portion 84 diverge away from top edge 86, topresent a more tapered profile. FIG. 13 shows a pad 88 having aposterior portion 90 and an anterior portion 92, wherein both portionsare substantially quadrilateral in shape, except for a sloping flatshoulder 94 at the juncture of portions 90, 92. FIG. 14 shows a pad 95that includes a posterior portion 96 of quadrilateral shape and ananterior portion 98 having upwardly converging side surfaces 100 and aflat leading edge 102.

While the pads shown in FIGS. 10-14 have posterior portions with flatbottom surfaces, the bottom surfaces may have other configurations.Thus, as shown in FIG. 15, the posterior portion P has an arcuate bottomsurface A, while in FIG. 16, the posterior portion P′ has convergingsurfaces C and an arcuate bottom edge B.

Further, alternative embodiments are shown in FIGS. 17 and 18. Thus, inFIG. 17, the non-bipartite pad 104 is of generally triangular crosssection, with a posterior portion 106 of large cross section and ananterior portion 108 of small cross section. The pad 104 has flat,converging surfaces 110, a slightly curved bottom surface 112, roundedbottom edges 114 and a rounded leading edge 116. The pad 118 shown inFIG. 18 is similar to pad 106, except that the anterior portion 120 istransversely constricted and provides a linear juncture J betweenposterior portion 122 and anterior portion 120. This is an example of abipartite pad that has major and minor portions.

FIG. 19 shows pad 124, which includes a posterior portion 126 ofsubstantially hexagonal cross section and a transversely constrictedanterior portion 128 with a rounded leading edge 130. The surfaces ofposterior portion 126 are flat and edges thereof may be rounded.

FIG. 20 shows pad 132, which includes a posterior major portion 134defined by opposed convergent flat surfaces 136 and a slightly roundedbottom surface 138; while anterior minor portion 140 is of a triangularcross section.

The pads may be suitably tapered in a longitudinal direction transverseto the AP direction. Thus pad 142, as shown in FIG. 21, has its anteriorportion 144 and posterior portion 146 tapered in respect of both thelongitudinal and transverse axes thereof; whereas in pad 148, as shownin FIG. 22, anterior portion 150 and posterior portion 152 are taperedlongitudinally only.

FIG. 23 shows yet another embodiment of the pad 154, in which theanterior portion 156 and posterior portion 158 are substantially ovoidin cross-section, with the transverse width of anterior portion 156 muchless than the transverse width of posterior portion 158.

The pads may be further modified, as shown in FIGS. 24-26. Thus, asshown in FIG. 24, the pad 160 has its posterior portion 162 sloped atone end as at 164, to make the pad conform to the anatomy of the user.Alternatively, as shown in FIG. 25, the pad 166 may be sloped at bothopposite ends 168, 168′. Alternatively, as shown in FIG. 26, pad 170 hasits posterior portion 172 sloped at opposite ends in a convergentconfiguration. If desired, in the foregoing embodiments, the anteriorportions of the pads may also be sloped to converge toward one another.FIG. 27 shows an embodiment of a pad 174 that has an anterior portion176 and posterior portion 177. The posterior portion 177 is formed witha longitudinal groove 178 of normally triangular section, forming wings180. When the pad 174 is inserted into the interlabial space, as shownin FIG. 28, the wings 180 are resiliently urged toward each other andbear against the labia majora, thereby increasing the retention of thepad within the interlabial space.

The various forms of pads set forth above may also include the groove inthe anterior portions thereof. The pads set forth above which haveopposed flat surfaces (e.g. FIGS. 11-14), are particularly adapted toconform to the medial surfaces of labia majora, for retention and cellcollection. Such embodiments that have slender projecting anteriorportions can also be configured such that the anterior projectioninserts into the vaginal opening, to further enhance retention of thepad.

Although some of the pads have been shown to taper longitudinally fromone end to the other end, they may also taper from a central portion tothe opposite ends thereof. Thus, while the pad may be of uniform crosssection throughout its length, it may also have a tapered form. In thisconfiguration, no string or other removal aid is required, and the padcan be removed manually, such as with a gentle tap.

Another embodiment of the interlabial pad 182 is shown in FIG. 29, inwhich the pad 182 has a posterior portion 184 and anterior portion 186,each having a cross section that defines a portion of a circle. Each ofthe posterior and anterior portions is a portion of a sphere that issymmetric in all directions with respect to a center point, and has aconstant radius. For example, posterior portion 184 is symmetric withrespect to center C1, while anterior portion 186 is symmetric withrespect to center C2.

FIG. 30 shows yet another embodiment of a pad 188 having merged portions190, 192 which are of part elliptical cross section; the portion 190having major and minor axes somewhat larger then those of portion 192,which also lends itself to easy insertion and removal. Portion 190 issymmetric in all directions with respect to perpendicular planes ofsymmetry, one of which is shown as P in FIG. 30. In this embodiment, thepad is not elongated in any direction, although in other embodimentslongitudinal elongation is possible.

The pad 194, shown in FIG. 31, is of an elliptical cross section. Thisembodiment lacks a major portion and a minor portion, and instead has across-section that is completely symmetric with respect to ananterior-posterior plane AP. In use, pad 194 is inserted along the APaxis into the interlabial space (either narrowed end of the pad can bethe leading edge of insertion).

The pad 200 shown in FIG. 32 is an elongated version of the pad in FIG.30, which has a more spherical configuration. Pad 200 in FIG. 32 isinitially of a round cross section, but is formed into a larger andsmaller portion by using a mechanical binding agent, such as thread orheat welding, similar to that described in Gerstenburger (U.S. Pat. No.5,575,047). Alternatively, it can be sewn along the junction between thetwo portions with biodegradable thread, so that the pad is completelybiodegradable, and can be flushed down a toilet. Biodegradable pads canbe made by any method, such as those disclosed in U.S. Pat. No.5,575,047, which is incorporated herein by reference.

In certain examples, the absorbent pads are additionally (oralternatively) impregnated with selected scents, to provide a soothingand pleasant odor. In one embodiment, the pad is impregnated with cellpreservation agents in the anterior (or minor) portion only (that fitsclosest to the vaginal opening), or in the posterior portion only. Inother embodiments, both the anterior (or minor) and posterior (or major)portions are impregnated with cell preservation agents. Alternatively,the anterior portion may be impregnated with medication, and theposterior portion is impregnated with an agent, or vice versa. Theseinclude, but are not limited to, hexamidine or zinc oxide (ZnO).

In one embodiment, the pad includes a groove in the anterior orposterior portion, and the scent, preservative, or another agent isadded within the groove or impregnated in the pad adjacent to thegroove. However, in other embodiments an agent is introduced into thepad by applying it as a liquid or powder to the pad. Thus, active agentscan be introduced on to the surface of the pad, impregnated throughoutit, or introduced into superficial regions of the pad, or parts of it.

FIG. 33 shows a cross-section of an embodiment of an elongated absorbentpad 200 that has been modified to carry agents for cell preservation.The principles of the elongated embodiment could, however, be adapted tothe non-elongated embodiments of the type shown in FIG. 29. In theembodiment shown in FIG. 33, pad 200 includes posterior portion 202 andanterior portion 204, which is formed with a groove 206 extendinglongitudinally along the top of anterior pad portion 204. The groove isprefilled with a material 208, for example, with an ointment,preservatives, lubricants, buffers and the like. Placing the interlabialpad in the interlabial space, with anterior portion 204 adjacent to thevaginal orifice and urethral orifice, causes the normal transverseconstriction of the pad 200 (and particularly compression of anteriorportion 204) to dispense materials, which have a suitable viscosity, tothe interlabial space.

It has been found that the curvilinear surface portions and thenon-uniform cross sections of the several pads shown herein are highlyeffective in positioning the pad in the interlabial space and retainingit in place. Further, there is no tendency to uncomfortably force thelabia majora apart or to exert undue pressure against their wallportions.

FIG. 34 shows a particular embodiment wherein an interlabial pad isformed of a polypropylene or polyester non-woven fabric with a rayonsliver core. As shown in FIG. 30, the interlabial pad has an overalllength L of about 15 to about 75 mm, and an overall height H of about 19to about 22 mm. Of the overall height of the interlabial pad, theanterior portion AP of the interlabial pad has a height APH of about 4to about 7 mm. The posterior portion PP of the interlabial pad has aheight PPH of about 12 to about 18 mm. In addition, posterior portion PPof the pad has a width PPW of about 8 to about 10 mm. Anterior portionAP has a width APW less than width PPW of posterior portion PP of thepad. In one specific, non-limiting example, width PPW of posteriorportion PP of the interlabial pad is from about 4 to about 7 mm. Theposterior portion PP of the pad is demarcated from anterior portion APof the pad by stitching S. In one specific, non-limiting example thestitching is standard 401 chain stitch of about 8-10 SPI.

In particular embodiments, the pad is formed of a soft absorptivematerial such as rayon, cellulose, cotton, or another suitable naturalor synthetic fiber or sheeting. In one embodiment the pad is flushable,and can be made of biodegradable material. In some embodiments, theabsorbent medium comprises an inner core and an outer covering, theouter covering having a visible matrix of pores of sufficient size toallow cells to enter the pores. The pad may be made as described in U.S.Pat. No. 5,575,047, herein incorporated by reference.

While interlabial pads are of use, they are not the only means of cellcollection. In some embodiments, a device placed intravaginally collectsfetal cells and/or components of fetal cells. Devices can be used suchas those disclosed in U.S. Pat. No. 6,174,293 and U.S. Pat. No.5,725,841, which is incorporated herein by reference. In addition,absorbent medium in the form of a tampon can be used, such as thosedisclosed, for example, in U.S. Pat. No. 7,713,253; U.S. Pat. No.7,341,737; U.S. Pat. No. 7,091,395; U.S. Pat. No. 6,743,212; or U.S.Pat. No. 6,155,990, which are incorporated herein by reference. Infurther embodiments, sanitary napkins or panty-liners can be utilizedfor the collection of fetal cells. These include a variety of sanitarynapkins that are commercially available, such as, but not limited to,those produced by UNICHARM®.

III. SEPARATION OF MATERNAL AND FETAL CELLS AND CELLULAR COMPONENTS

In some embodiments, after fluid and cells are collected on an absorbentmedium, such as an interlabial pad, sanitary napkin, tampon orpanty-liner, the cells and/or cellular components can be extracted fromthe absorbent medium. This can involve placing the absorbent medium intoa liquid that retains the viability of the cells, such as a tissueculture medium or physiological buffer, such as a buffer with a pH ofabout 7 to about 7.6, such as about 7.2 to about 7.4, such as about 7.2,about 7.3 or about 7.4. The release of the cells and/or cellularcomponents from the absorbent medium can include shaking, vibration,light sonication, or any method which allows the release of the cellsand/or cellular components but retains cell viability. In onenon-limiting example, the absorbent medium is agitated for about 1 toabout 30 minutes, such as for about 2 to about 5 minutes, in thepresence of a physiological buffer, such as phosphate buffered saline orDulbecco's modified Eagle's medium.

Following release of cells and/or cellular components, the tissueculture medium or physiological buffer can then be centrifuged to form apellet of cellular material. This cellular material can be resuspendedat a desired concentration in an additional portion of a medium orphysiological buffer. In some embodiments, a cell suspension is producedfrom the cell pellet for further propagation of the cells. In otherembodiments, cellular components are extracted from the cellularmaterial.

In further embodiments, the supernatant is collected followingcentrifugation to form a pellet of cellular material. This supernatantincludes cellular components, such as DNA, RNA, proteins and/or lipids.The cellular components then can be isolated using methods known tothose of skill in the art. For example, the extraction of fetal DNA andRNA from maternal samples is disclosed in U.S. Published PatentApplication No. 20120108460; these methods are also of use with regardto the supernatant. For example, genomic DNA can be isolated using aQIAGEN® Kit for purification of DNA from blood cells, following themanufacturer's instructions (for example, QIAmp DNA Blood Midi Kit,Catalog number 51183).

Once obtained, the sample of cells and/or cellular components can bestored at room temperature until use. In other embodiments, the samplecan be stored at 0 to 4° C. until use. The sample can be transportedand/or stored at 4° C. For long-term storage, the sample can be storedin at −80° C. In one embodiment, when cells are collected and/or storedthe medium comprises serum such as bovine calf serum or human serum. Insome examples, GIBCO® AMNIOMAXII™, GIBCO® AMNIOMAX™ C-100, or GIBCO®keratinocyte-serum free media supplemented serum can be used. In afurther embodiment, the medium is degassed with nitrogen to reduceoxidative stress to the samples.

In some embodiments, when cells are to be analyzed, fetal cells areseparated from maternal cells, in order to isolate the fetal cells. Thiscan be accomplished by a variety of methods including, for example,fluorescence activated cells sorting (FACS). Fetal cells can bepositively and/or maternal cells can be negatively selected, using avariety of techniques well known in the art, including cell sorting,especially FACS, by using an affinity reagent bound to a substrate(e.g., a plastic surface, as in panning), or by using an affinityreagent bound to a solid phase particle which can be isolated on thebasis of the properties of the solid phase particles for example beads(e.g., colored latex beads or magnetic particles). The procedure usedwill depend on whether maternal or fetal cells are being selected andhow the cells have been labeled. For selection of cells by cell sorting,the cells are labeled directly or indirectly with a substance which canbe detected by a cell sorter, preferably a dye. The dye can be afluorescent dye. A large number of different dyes are known in the art,including fluorescein, rhodamine, Texas red, phycoerythrin, and thelike. Any detectable substance, which has the appropriatecharacteristics for the cell sorter, may be used (e.g., in the case of afluorescent dye, a dye which can be excited by the sorter's lightsource, and an emission spectra which can be detected by the cellsorter's detectors).

For the selection of cells from a sample using solid-phase particles,any particle with the desired properties may be utilized. For example,large particles (e.g., greater than about 90-100 μm in diameter) may beused to facilitate sedimentation. Preferably, the particles are“magnetic particles” (i.e., particles which can be collected using amagnetic field). Typically, maternal cells labeled with the magneticprobe are passed through a column, held within a magnetic field. Labeledmaternal cells are retained on the column (held by the magnetic field),while unlabeled fetal cells pass straight through and are eluted at theother end. Magnetic particles are now commonly available from a varietyof manufacturers including Dynal Biotech (Oslo, Norway) and MiltenyiBiotech GmbH (Germany). An example of magnetic activated cell sorting(MACS) is provided in U.S. Pat. No. 4,675,286. Laser-capturemicro-dissection can also be used to select labeled cells. Methods ofusing laser-capture micro-dissection are known in the art (see, forexample, U.S. Published Patent Application No. 2003/0227611).

In flow cytometry, a beam of laser light is projected through a liquidstream that contains cells, or other particles, which when struck by thefocused light give out signals which are picked up by detectors. Thesesignals are then converted for computer storage and data analysis, andcan provide information about various cellular properties. In someembodiments, forward scatter data can be used to select and/or enrichfetal cells, either multinucleated and/or non-multinucleated, based oncell size. For example, when a laser hits the cell, the larger the cellthe more photons of light it scatters. By measuring the light scatteredon the side of a cell furthest from where the laser hits the cell, ameasure of cell size can be obtained, and fetal cells of a particularsize can be isolated.

Many larger flow cytometers are also “cell sorters”, such asfluorescence-activated cell sorters (FACS), and are instruments, whichhave the ability to selectively deposit cells from particularpopulations into tubes, or other collection vessels. In an embodiment,the cells are isolated using FACS. This procedure is well known in theart and described by, for example, Melamed, et al. Flow Cytometry andSorting Wiley-Liss, Inc., New York, N.Y. (1990); Shapiro Practical FlowCytometry, 4 ed, Wiley-Liss, Hoboken, N.J. (2003); and Robinson et al.Handbook of Flow Cytometry Methods Wiley-Liss, New York, N.Y. (1993);Harkins and Galbraith (1987) and U.S. Pat. No. 4,765,737.

In order to sort cells, the instrument's electronics interprets thesignals collected for each cell as it is interrogated by the laser beamand compares the signal with sorting criteria set on the computer. Ifthe cell meets the required criteria, an electrical charge is applied tothe liquid stream, which is being accurately broken into dropletscontaining the cells. This charge is applied to the stream at theprecise moment the cell of interest is about to break off from thestream, then removed when the charged droplet has broken from thestream. As the droplets fall, they pass between two metal plates, whichare strongly positively or negatively charged. Charged droplets getdrawn towards the metal plate of the opposite polarity, and deposited inthe collection vessel, or onto a microscope slide, for furtherexamination.

The cells can automatically be deposited in collection vessels as singlecells or as a plurality of cells, such as using a laser, for example anargon laser (488 nm) and for example with a Flow Cytometer fitted withan Autoclone unit (Coulter EPICS Altra, Beckman-Coulter, Miami, Fla.,USA). Other examples of suitable FACS machines useful for the methods ofthe invention include, but are not limited to, MOFLO® High-speed cellsorter (Dako-Cytomation Ltd), FACS ARIA® (Becton Dickinson), ALTRA®Hyper sort (Beckman Coulter) and CYFLOW® sorting system (Partec GmbH).

Fetal and maternal cells can be separated based on the expression ofgenes in the major histocompatibility complex (MHC). The MHC includes atleast three classes of genes. Class I and II genes encode antigensexpressed on the cell surface, whilst class III genes encode severalcomponents of the complement system. Classes I and II antigens areglycoproteins that present peptides to T lymphocytes. Human MHCmolecules are also known in the art as Human Leukocyte Antigens (HLA).Thus, the terms “HLA” and “MHC” are often used interchangeably.

Human and murine class I molecules are heterodimers, consisting of aheavy alpha chain (45 kD) and a light chain, beta-2-globulin (12 kD).Class I molecules are found on most, if not all, nucleated cells. Thealpha chain can be divided into three extracellular domains, alpha1,alpha2 and alpha3, in addition to the transmembranous and cytoplasmicdomains. The alpha3 domain is highly conserved, as isbeta-2-microglobulin. Both alpha3 domain and beta-2-microglobulin arehomologous to the CH3 domain of human immunoglobulin. There are 3 classI loci (B,C,A) in the short arm of human chromosome 6, and 4 loci (K,D(L), Qa, Tla) in murine chromosome 17. These loci are highlypolymorphic. The variable residues are clustered in 7 subsequences, 3 inalpha1 domain and 4 in alpha2 domain. There are three major human classII loci (HLA-DR, HLA-DO, HLA-DP). All class II beta chains arepolymorphic. The human HLA-DQ alpha chain is also polymorphic.

Agents, such as an antibody that specifically bind an MHC molecule, canbe used to isolate fetal and maternal cells. Generally, antibodies areof use that specifically bind an extracellular portion of the MHCmolecule. In this manner, the method can be used to enrich live fetalcells. Furthermore, an additional step of ensuring that the agent passesthrough the cell membrane (for example having to fix and permeabilizethe cell) is not required. In one embodiment, an antibody thatspecifically binds HLA-A, HLA-B and HLA-C molecule is utilized. In oneembodiment, an antibody is utilized that specifically binds HLA-A orHLA-B molecules. More than one antibody can be used, wherein eachantibody specifically binds a different classes or sub-classes of MHCmolecules. A “sub-class” of a MHC molecule is a distinct type of MHCmolecules of a particular class.

Thus, the method can include i) contacting the cells with an antibodythat specifically binds at least one MHC molecule, and ii) removingcells bound by the agent. More than one antibody, which specificallybinds an MHC molecule can be used. For example, in an embodiment, themethod comprises contacting the cells with i) an antibody thatspecifically binds a Class I MHC molecule, and ii) an antibody thatspecifically binds at least one Class II MHC molecule to separate fetalcells.

There are maternal cell specific markers that are not expressed on atleast the majority of fetal cells. Those skilled in the art are awarethat the types of nucleated maternal cells in maternal blood include Bcells, T cells, monocytes, macrophages dendritic cells and stem cells,each characterized by a specific set of surface markers that can betargeted for depletion. Examples of non-MHC molecules, which can betargeted to possibly further deplete the sample of maternal cellsinclude, but are not limited to, CD3, CD4, CD8, CD10, CD14, CD15, CD45,CD56. For example, magnetic beads can be produced which have bothanti-MHC and anti-CD45 antibodies attached the bead, which can be thenutilized for cell separation. Examples of maternal cells that may bedepleted include, but are not limited to, vaginal epithelial cells,cervical epithelial cells, endometrial cells, maternal endothelialcells, maternal placental cells, polymorphs and mesenchymal cells of theplacental villi.

Fetal cells can be positively selected by using agents, such asantibodies which specifically bind molecules, typically proteins, whichare not significantly produced by maternal cells in the sample. Examplesof fetal cell markers include, but are not limited to, molecules thatare expressed by syncytiotrophoblasts and/or cytotrophoblasts, but isnot expressed by maternal cells. Examples include, but are not limitedto, NDOG1 (AbCam, GeneTex, Serotec), NDOG2, Human Chorionic Gonadotropin(Calbiochem), MCP/cd46 (trophoblast/lymphocyte cross-reactive protein)(Abnova), TPBG (Trophoblast glycoprotein) (Abnova), GCSF receptor, ADFP(Adipose Differentiation Related Protein) (GenWay), Apolipoprotein H(AbCam), Placental Alkaline Phosphatase (AbCam), CXCR6 (Chemokinereceptor 6) (R&D Systems), HLA-G (AbCam), CHL1 (extravillouscytotrophoblast antigen) (Abnova), Cytokeratin 7 (AbCam), Cytokeratin 8(AbCam), Cytokeratin 18 (AbCam), FAS-Associated Phosphatase-1 (Leica),Folate Binding Protein (AbCam), FD0161G, Glucose Transporter GLUT3,H315, H316, HAI-1 (Hepatocyte growth factor activator protein-1(EBioscience)), Human Placental Lactogen (Serotec), Id-1, Id-2, IBSP(Integrin Binding SialoProtein), MCSF-Receptor, MNF116, OKT9,plasminogen activator inhibitor 1 (AbCam), PLP-A (prolactin likeproteins A) (Millipore Corporation), PLP-B (prolactin like proteins B),PLP-C(prolactin like proteins C), PLP-D (prolactin like proteins D),PLP-F (prolactin like proteins F), PLP-L (prolactin like proteins L),PLP-M (prolactin like proteins M), PLP-N(prolactin like proteins N),SP-1 (trophoblast specific beta 1 glycoprotein) (AbCam, BD Pharmingen),SSEA (Stage Specific Embryonic Antigen) (Novus Biologicals), TA1, TA2,Tfeb, Troma1, Trop1 (EBioscience) and Trop2, URO-4 (Adenosine DeaminaseBinding Protein [ABM]) (Covance). Fetal cells can also be isolated basedon the expression of a combination of any two or more thereof. In someembodiments, the fetal cells are selected using an agent which bindssyncytiotrophoblasts such as a monoclonal antibody which binds NDOG1. Inother embodiments, the fetal cells are selected using combinations ofagents which bind to villous syncytiotrophoblasts, villouscytotrophoblasts and extra villous cytotrophoblasts. For example, thecombination of agents may include an agent which binds NDOG1(Syncytiotrophoblasts), an agent which binds SP-1 (VillousCytotrophoblasts and villous syncytiotrophoblasts), and an agent whichbinds HLA-G (ExtraVillous Cytotrophoblasts).

Once fetal cells are separated, they can be propagated in culture usingmethods known in the art. For example for propagating embryonic stem(ES) cells, ES cell medium can be used. This medium is 80% Dulbecco'smodified Eagle's medium (DMEM; no pyruvate, high glucose formulation,GIBCO® BRL), with 20% fetal bovine serum (FBS; Hyclone), 0.1 mMβ-mercaptoethanol (Sigma), 1% non-essential amino acid stock (GIBCO®BRL). Generally, primate ES cells are isolated on a confluent layer ofmurine embryonic fibroblast in the presence of ES cell medium. In oneexample, embryonic fibroblasts are obtained from 12 day old fetuses fromoutbred mice (such as CF1, available from SASCO), but other strains maybe used as an alternative. Tissue culture dishes treated with 0.1%gelatin (type I; Sigma) can be utilized. Distinguishing features of EScells, as compared to the committed “multipotential” stem cells presentin adults, include the capacity of ES cells to maintain anundifferentiated state indefinitely in culture, and the potential thatES cells have to develop into every different cell types. Dissociatedcells are re-plated on embryonic feeder layers in fresh ES medium, andobserved for colony formation. Colonies demonstrating ES-like morphologyare individually selected, and split again as described above. TheES-like morphology is defined as compact colonies having a high nucleusto cytoplasm ratio and prominent nucleoli. Resulting ES cells are thenroutinely split manual disaggregation every 5-7 days as the culturesbecome dense. Early passage cells are also frozen and stored in liquidnitrogen. Cell lines can be karyotyped with a standard G-bandingtechnique and compared to published karyotypes for the primate species.

Of course, a variety of cell culture methods are available in the artand can be used to propagate embryonic cells of interest. In someembodiments, the sample is seeded on the feeder layer for stem cellculture under sterile conditions. Mycoplasma and other contaminationscan be examined. ESC-like cells can be examined by an Applied StemCell,Inc. ESC/iPSC characterization kit (immunofluorescence). Thus, theexpression of OCT4, SOX2, SSEA4, TRA-1-60, and TRA 1-81 is examined.

Addition methods for isolating fetal cells from liquid samples are knownand, in some embodiments, are used in the disclosed methods. Forexample, additional methods for isolating fetal cells include thosedisclosed in Patent Publications US20030013123, WO1990006509,WO1991007660, WO1995026417, WO1998002528, WO1998018005, WO2000071987,WO2003042405, WO2004076653, WO2005100401, WO2007106838, WO2007112281,and WO2009039507, each of which is incorporated by reference herein inits entirety.

IV. DETECTION OF CHROMOSOMAL ABNORMALITIES AND DIAGNOSTIC TESTING

In some embodiments, diagnostic testing is performed on cells and/orcellular components that are collected using the methods disclosedherein. The diagnostic test can detect, for example, the presence orabsence of a cell type (e.g. see U.S. Pat. No. 5,124,252 and U.S. Pat.No. 5,965,375, each of which is incorporated by reference herein in itsentirety), a protein (e.g. see U.S. Pat. No. 5,190,881 and U.S. Pat. No.5,661,010, each of which is incorporated by reference herein in itsentirety), a lipid, or a nucleic acid (e.g., see U.S. Pat. No. 5,538,851and U.S. Pat. No. 5,459,034, each of which is incorporated by referenceherein in its entirety).

In several embodiments, the diagnostic test is performed by a thirdparty.

In several non-limiting examples, the cells or cellular components areanalyzed to determine the response or absence of a genomic or epigeneticcharacteristic associated with a biological outcome (e.g., a phenotypeexhibited by the subject from which the cells or cellular components arederived), such as the presence or absence of a Y chromosome, or thepresence or absence of an aneuploidy (e.g. the presence or absence ofmore than two copies of chromosome 21).

In some specific, non-limiting examples, the cells or cellularcomponents are analyzed to detect the presence of a Y chromosome, todetermine if the fetus is male. In other embodiments the cells orcellular components are analyzed to detect chromosomal abnormalities inthe fetus. These methods can include the separation of fetal cells, butin some embodiments fetal cells need not be separated from maternalcells.

The methods can include the isolation of cellular components such asDNA, RNA, proteins or lipids. The cellular component that is analyzedcan be genetic material, including RNA, nuclear DNA or mitochondrialDNA. However, at least in some instances it may be informative toanalyze RNA or protein. Furthermore, the DNA may encode a gene, or mayencode a functional RNA which is not translated, or the DNA analyzed mayeven be an informative non-transcribed sequence or marker.

Methods for isolation of these components are known in the art. In someembodiments, the cellular components are collected directly from thesample. Cellular components can be extracted from the fetal cellspresent in the sample. Thus, in some embodiments, fetal cellularcomponents, such as fetal DNA, RNA, protein and/or lipids are separatedfrom the maternal cellular components, such as maternal DNA, RNA,proteins and/or lipids. However, for some analysis, the separation offetal cellular components from maternal cellular components is notrequired. In some embodiments, a mixture of maternal and fetal cellularcomponents are isolated from the absorbent medium, and these cellularcomponents are then subjected to diagnostic testing for the fetus. Inone specific, non-limiting example, the cellular components are testedfor the presence of a Y chromosome, to determine if the fetus is male.In other embodiments the cellular components are analyzed to detectchromosomal abnormalities in the fetus.

DNA can be extracted and concentrated by known methods, includingcentrifugation and various enzyme inhibitors. In some embodiments, theDNA is bound to a selective membrane (e.g., silica) to separate it fromcontaminants. Fetal DNA can be hypomethylated relative to adult DNAreflecting transcriptional silencing of specific genes expressed earlyin development. One means of generating fetal-specific PCR products isto identify loci that are unmethylated in fetal DNA and methylated inadult/maternal DNA. Another means to detect fetal-specific DNA is toidentify loci that are methylated in fetal DNA and unmethylated inadult/maternal DNA. Loci of this type are differentially reactive withbisulfite such that unmethylated Cs in DNA undergo oxidativedeamination, resulting in C to U transitions. Methylated Cs are notreactive with bisulfite, and consequently, are unaffected. Bisulfitetreatment of fetal and maternal DNA present in maternal serum willcreate primary sequence differences between fetal and maternal loci thatexhibit differential methylation. However, restriction enzymes thatdifferentially recognize and clear unmethylated DNA can also be used. Inother embodiments, the method for selective enrichment of fetal DNArequires the use of the methyl-CpG binding domain of human MBD2 protein,which is coupled to paramagnetic beads, for example DYNABEADS®280Streptavidin, via a biotin linker Without being bound by theory, thehigh affinity of the MBD-biotin protein for CpG-methylated DNA providesgreater sensitivity than antibody binding, while the use of theDYNABEADS® provides a simplified, streamlined workflow.

In one embodiment, chromosomal abnormalities are detected. This includesa gross abnormality in a chromosome or the number of chromosomes. Forexample, this includes detecting trisomy in chromosome 21 which isindicative of Down's syndrome, trisomy 18, trisomy 13, sex chromosomalabnormalities such as Klinefelter syndrome (47, XXY), XYY or Turner'ssyndrome, chromosome translocations and deletions, a small proportion ofDown's syndrome patients have translocation and chromosomal deletionsyndromes which include Pradar-Willi syndrome and Angelman syndrome,both of which involve deletions of part of chromosome 15, and thedetection of mutations (such as deletions, insertions, transitions,transversions and other mutations) in individual genes. Other types ofchromosomal problems also exist such as Fragile X syndrome, hemophilia,spinal muscular dystrophy, myotonic dystrophy, Menkes disease andneurofibromatosis, which can be detected by DNA analysis.

Genetic abnormalities such as a single nucleotide substitution,deletion, insertion, micro-deletion, micro-insertion, short deletion,short insertion, multinucleotide substitution, and abnormal DNAmethylation and loss of imprint (LOI) can be detected. Such a geneticabnormality can be related to an inherited genetic disease such as asingle-gene disorder (e.g., cystic fibrosis, Canavan, Tay-Sachs disease,Gaucher disease, Familial Dysautonomia, Niemann-Pick disease, Fanconianemia, Ataxia telengectasia, Bloom syndrome, Familial Mediterraneanfever (FMF), X-linked spondyloepiphyseal dysplasia tarda, factor XI), animprinting disorder [e.g., Angelman Syndrome, Prader-Willi Syndrome,Beckwith-Wiedemann syndrome, Myoclonus-dystonia syndrome (MDS)], or topredisposition to various diseases (e.g., mutations in the BRCA1 andBRCA2 genes). Other genetic disorders which can be detected by DNAanalysis are known such as thalassaemia, Duchenne muscular dystrophy,connexin 26, congenital adrenal hypoplasia, X-linked hydrocephalus,ornithine transcarbamylase deficiency, Huntington's disease,mitochondrial disorder, mucopolysaccharidosis I or IV, Nome's disease,Rett syndrome, Smith-Lemli Optiz syndrome, 21-hydroxylase deficiency orholocarboxylase synthetase deficiency, diastrophic dysplasia,galactosialidosis, gangliosidosis, hereditary sensory neuropathy,hypogammaglobulinaemia, hypophosphatasia, Leigh's syndrome,aspartylglucosaminuria, metachromatic leukodystrophy Wilson's disease,steroid sulfatase deficiency, X-linked adrenoleukodystrophy,phosphorylase kinase deficiency (Type VI glycogen storage disease) anddebranching enzyme deficiency (Type III glycogen storage disease). Theseand other genetic diseases are mentioned in The Metabolic and MolecularBasis of Inherited Disease, 8th Edition, Volumes I, II, III and IV,Scriver, C. R. et al. (eds), McGraw Hill, 2001. Clearly, any geneticdisease where the gene has been cloned and mutations detected can beanalyzed.

The methods can also be used to determine the sex of the fetus. Forexample, staining of the isolated fetal nuclei with a Y chromosomespecific marker will indicate that the fetus is male, whereas the lackof staining will indicate that the fetus is female.

In yet other embodiments, the methods described herein can be used forpaternity testing. Where the paternity of a child is disputed, theprocedures of the invention enable this issue to be resolved early onduring pregnancy by testing fetal cells. Many procedures have beendescribed for parentage testing which rely on the analysis of suitablepolymorphic markers. Polymorphic markers include any nucleic acid change(e.g., substitution, deletion, insertion, inversion), variable number oftandem repeats (VNTR), short tandem repeats (STR), minisatellite variantrepeats (MVR) and the like. Typically, parentage testing involves DNAfingerprinting targeting informative repeat regions, or the analysis ofhighly polymorphic regions of the genome such as HLA loci.

Chromosomal abnormalities, either in structure or number, can bedetected by karyotyping. Karyotyping analysis is generally performed onnuclei which have been arrested during mitosis by the addition of amitotic spindle inhibitor such as colchicine. In some embodiments, aGiemsa-stained chromosome spread is prepared, allowing analysis ofchromosome number as well as detection of chromosomal translocations.

The genetic assays can involve any suitable method for identifyingmutations or polymorphisms in the fetal DNA, such as: sequencing of theDNA at one or more of the relevant positions; differential hybridizationof an oligonucleotide probe designed to hybridize at the relevantpositions of either the wild-type or mutant sequence; denaturing gelelectrophoresis following digestion with an appropriate restrictionenzyme, preferably following amplification of the relevant DNA regions;S1 nuclease sequence analysis; non-denaturing gel electrophoresis,preferably following amplification of the relevant DNA regions;conventional RFLP (restriction fragment length polymorphism) assays;selective DNA amplification using oligonucleotides which are matched forthe wild-type sequence and unmatched for the mutant sequence or viceversa; or the selective introduction of a restriction site using a PCR(or similar) primer matched for the wild-type or mutant genotype,followed by a restriction digest. The assay may be indirect, such thatit is capable of detecting a mutation at another position or gene whichis known to be linked to one or more of the mutant positions. The probesand primers can be fragments of DNA isolated from nature or may besynthetic. A non-denaturing gel can be used to detect differing lengthsof fragments resulting from digestion with an appropriate restrictionenzyme. The DNA is usually amplified before digestion, for example usingthe polymerase chain reaction (PCR) method and modifications thereof.

Amplification of fetal DNA can be achieved by the established PCRmethods or by developments thereof or alternatives such as quantitativePCR, quantitative fluorescent PCR (QF-PCR), multiplex ligation dependentprobe amplification, digital PCR, real time PCR (RT-PCR), single nucleiPCR, restriction fragment length polymorphism PCR (PCR-RFLP),PCR-RFLP/RT-PCR-RFLP, hot start PCR, nested PCR, in situ polonony PCR,in situ rolling circle amplification (RCA), bridge PCR, picotiter PCRand emulsion PCR. Other suitable amplification methods include theligase chain reaction (LCR), transcription amplification, self-sustainedsequence replication, selective amplification of target polynucleotidesequences, consensus sequence primed polymerase chain reaction (CP-PCR),arbitrarily primed polymerase chain reaction (AP-PCR), degenerateoligonucleotide-primed PCR (DOP-PCR) and nucleic acid based sequenceamplification (NABSA). Other amplification methods that can be usedherein include those described in U.S. Pat. No. 5,242,794; U.S. Pat. No.5,494,810; U.S. Pat. No. 4,988,617; and U.S. Pat. No. 6,582,938.

Generally, an “appropriate restriction enzyme” will recognize and cutthe wild-type sequence and not the mutated sequence or vice versa. Thesequence which is recognized and cut by the restriction enzyme (or not,as the case may be) can be present as a consequence of the mutation orit can be introduced into the normal or mutant allele using mismatchedoligonucleotides in the PCR reaction. It is convenient if the enzymecuts DNA only infrequently, in other words if it recognizes a sequencewhich occurs only rarely. In another method, a pair of PCR primers areused which hybridize to either the wild-type genotype or the mutantgenotype but not both. Whether amplified DNA is produced will thenindicate the wild-type or mutant genotype (and hence phenotype).

Another method employs similar PCR primers but, as well as hybridizingto only one of the wild-type or mutant sequences, they introduce arestriction site which is not otherwise there in either the wild-type ormutant sequences. In order to facilitate subsequent cloning of amplifiedsequences, primers may have restriction enzyme sites appended to their5′ ends. Thus, all nucleotides of the primers are derived from the genesequence of interest or sequences adjacent to that gene except the fewnucleotides necessary to form a restriction enzyme site. Such enzymesand sites are well known in the art. The primers themselves can besynthesized using techniques which are well known in the art. Generally,the primers can be made using synthesizing machines which arecommercially available.

PCR techniques that utilize fluorescent dyes may also be used to detectgenetic defects in DNA from fetal cells isolated by the methodsdisclosed herein. Fluorescent dyes can be used to detect specific PCRamplified double stranded DNA product (e.g. ethidium bromide, or SYBRGreen I). The 5′ nuclease (TTAQMAN®) assay can be used which utilizes aspecially constructed primer whose fluorescence is quenched until it isreleased by the nuclease activity of the Taq DNA polymerase duringextension of the PCR product. Assays based on Molecular Beacontechnology can be used which rely on a specially constructedoligonucleotide that when self-hybridized quenches fluorescence(fluorescent dye and quencher molecule are adjacent). Upon hybridizationto a specific amplified PCR product, fluorescence is increased due toseparation of the quencher from the fluorescent molecule. Assays basedon Amplifluor (Intergen) technology can be used which utilize speciallyprepared primers, where again fluorescence is quenched due toself-hybridization. In this case, fluorescence is released during PCRamplification by extension through the primer sequence, which results inthe separation of fluorescent and quencher molecules. Assays that relyon an increase in fluorescence resonance energy transfer can be usedwhich utilize two specially designed adjacent primers, which havedifferent fluorochromes on their ends. When these primers anneal to aspecific PCR amplified product, the two fluorochromes are broughttogether. The excitation of one fluorochrome results in an increase influorescence of the other fluorochrome.

The acronym “FISH” references a technique that uses chromophore tags(fluorophores) that emit a secondary signal if illuminated with anexcitation light to detect a chromosomal structure. FISH usesfluorescent probes which bind only to those parts of the chromosome withwhich they show a high degree of sequence similarity. Such tags may bedirected to specific chromosomes and specific chromosome regions. Theprobe has to be long enough to hybridize specifically to its target (andnot to similar sequences in the genome), but not too large to impede thehybridization process, and it should be tagged directly withfluorophores. This can be done in various ways, for example nicktranslation or PCR using tagged nucleotides. If signal amplification isnecessary to exceed the detection threshold of the microscope (whichdepends on many factors such as probe labeling efficiency, the kind ofprobe and the fluorescent dye), secondary fluorescent tagged antibodiesor streptavidin are bound to the tag molecules, thus amplifying thesignal.

Any known sequencing methods can be used to analyze DNA. Such sequencingmethods provide sequence information at the single nucleotide level andthus allow for the detection of mutations and other abnormalities thatoccur in one genotype in the biological sample, but not the other.

EXAMPLES

The following examples are provided to illustrate particular features ofcertain embodiments, but the scope of the claims should not be limitedto those features exemplified.

Example 1 Collection of Fetal DNA

For this study, interlabial pads (PADKIT®) were used for collection ofsamples containing the products of approximately 2-6 hours ofcervicovaginal discharge (maternal vaginal sample). Each PADKIT®contains two (2) interlabial pads (one for the sample, and one extra), aplastic glove, a collection vial containing a preservative solution,inside a transport tube, and a special self-addressed envelope. Thegoals were to determine if fetal DNA was present in a maternal vaginalsample, and comparing the performance of a new specimen collectionmethod with the ultrasound outcome in a targeted population of women,known to be pregnant with a male child. The design of this studyinvolved home-based sample collection, and included a blinded analysisof results developed from study samples. This study was designed tocompare maternal vaginal samples collected with the PADKIT® to othermethods of fetal sex determination by the identification of the “Y”chromosome within the maternal sample. The Y chromosome of the child isidentical to the Y chromosome of the father. Thus, the participants wereasked to abstain from all sexual intercourse during the period from 24hours prior to ultrasound screening, until the sample collection wascompleted, so that Y chromosomes from sperm will not contribute to DNAmeasurements for fetal cells. The study design is shown in FIG. 1.

Following enrollment, the study subject began the self-collection ofmaternal vaginal samples using the supplies provided in the PADKIT®.Each subject collected two vaginal PADKIT® samples over two days,beginning 1 day, but not more than 14 days after ultrasound sexdetermination. Each PADKIT® interlabial pad was kept in place 2-4 hours,one sample taken each day, after first morning void.

Maternal Vaginal Sample Collection:

-   -   1. Two (2) maternal vaginal samples were collected.    -   2. Subject collected one maternal vaginal sample each morning        for two days, beginning 1 day, but not more than 14 days, after        ultrasound testing.    -   3. A Specimen Collection Log entry was completed for each sample        by the study subject, and forwarded to the Study Coordinator.    -   4. A Vial Label, traceable to the Specimen Collection Log entry,        was completed and affixed to the collection vial for each sample        collected.    -   5. Specimens were forwarded to a US based laboratory, via US        mail.    -   6. At the completion of the Study, the residual fraction of all        samples were sent to, and stored at QuantRx for future        development work

Sample Handling/Processing/Analysis

-   1. The PADKIT® sample, in a vial, was forwarded to a US-based    laboratory for testing.-   2. The Lab received the sample, assigned a unique    accessioning/tracking number and recorded the information specified    on the Specimen Receipt Log.-   3. The Sample Processing/Interpretation Performed Included:    -   a) Elution of the cellular material from the interlabial pad    -   b) Removal of the interlabial pad from the vial.    -   c) Centrifugation of the vial.    -   d) Using the cell pellet for preparation of genomic tests    -   e) Evaluation of the tests.-   4. A description of findings was completed for each specimen.

Example 2 Clinical Study Study Objectives:

-   -   PADKIT® samples obtained from patients post ultrasound        identification of fetal sex (male)

Study Design:

-   -   Home-based sample collection was used to perform blinded testing        from study samples.

Study Sample:

-   -   Pregnant women, who have opted to identify the sex of their        fetus via ultrasound, were enrolled.    -   Ultrasound identification of a male fetus    -   Collect and mailed two (2) PADKIT® samples, and abstain from        intercourse from 24 hours prior to the Ultrasound screening        until the collection process is complete.

Results:

-   -   Ten (10) samples+two (2) controls have been tested    -   100% of the samples are positive for the “Y” chromosome    -   Controls are clearly negative for “Y” chromosome    -   All Study Samples are positive for “Y” chromosome    -   All Study Samples have been shipped 6,873 miles    -   FIGS. 2 and 3 show the study results wherein fetal (“Y”) DNA was        detected in the samples.    -   FIGS. 4 and 5 show the cytology results.

Sample Collection:

The subjects were provided with two (2) PADKIT® packets for collectingmaternal vaginal samples. Each PADKIT® contained the following:

-   -   Two (2) interlabial pads. One for your sample, Two extras if        needed    -   One (1) disposable glove        Subjects placed the first interlabial pad, and began collecting        the first sample within 24 hours of returning home from the        clinic. They were instructed not to start the study if they had        sexual intercourse since entering into the study protocol. They        placed the second interlabial pad and began collecting that        sample within 12 hours after retrieving the first sample and        placed it in the transport vial. They were instructed that they        were providing two maternal vaginal samples; this process will        take between 2 to 6 hours for each sample, and 4 hours was        considered optimal. There were also instructed to empty their        bladder completely; and not to urinate until after the sample        was taken.

Instructions for inserting and removing the interlabial pad were:

-   -   Stand or sit comfortably, with knees spread apart—allowing the        labial (vaginal) lips to open.    -   Hold the interlabial pad with the thumb and finger, and gently        press it between the vaginal lips.    -   When the knees are brought together and stand up, the vaginal        lips naturally folded around the interlabial pad, holding it in        place.    -   The interlabial pad was retained in place for 2 to 6 hours; with        4 hours considered optimal.    -   When the pad was removed, the opened sample vial was placed on a        counter, the used pad was placed in the vial, the lid was closed        and placed in a mailer for shipping to the test facility.

PADKIT® Sample QC Via Digital PAP Cytometry in a CLIA/CAP CertifiedLaboratory:

Upon harvest as a pellet, ½ of each PADKIT® sample was subjected todigital cytometry using standard HOLOGICS® Thin-prep technology and Papstaining performed at a CLIA-certified Cytometry lab. These Thin-prepslides were then analyzed by APERIO® digital cytometry. Representativedata are shown in FIG. 5. Both standard light and digital cytometryconfirmed that all samples processed for this study showed ordinary cellmorphology, which as assessed by the Cytometry lab, wereindistinguishable relative to routine PAP stained cervical scrapes. SeeFIGS. 4-5.

In order to obtain the ability to detect signal from the male version ofthe Amelogenin (Amel-Y) marker gene in the presence of a 100 to 10,000fold excess of the very similar female version of Amelogenin on the Xchromosomes of the mother (Amel-X), Y-specific tandem PCR reactions wererun, followed by hybridization to the Y-Chip. Positive hybridizationsignals for the six Y-specific hybridization probes (y1-y6) were seenfor all samples except for non-pregnant control samples 22A,B and 25A,B.The data demonstrate good false positive signal behavior (no X-specificsignals for any sample) and no Y-specific signals on the X0X (female)controls 22A,b & 25A,B. (FIGS. 2-3)

1 uL of the retained PadKit cell suspension in Tris not used forCytometry (typically 50 uL) was subjected to same sort of tandem PCRreactions described above for Amelogenin, but previously optimized forHLA-Typing at GMS. In these pilot HLA-Typing studies, the raw cellpellets from 12 anonymous participants were obtained via PADKIT®collection. They were subjected to HLA-Typing at the DRB1 locus. The 2°PCR amplicon product derived from the DRB1 amplification reactions wasanalyzed on an agarose gel, to generate the expected 250 bp DRB1 2°amplicon product (FIG. 6). This Cy-3 labeled amplicon was then used asthe target for standard HLA-Chip analysis in the 576 probe (12 well)format.

It was determined that the cells were viable for 48 hours in shipment.Cells remain viable for up to 5 years when refrigerated.

Example 3 Collection of Fetal Cells Using an Interlabial Pad

This example describes a method for collecting and isolating fetal cellsfrom a pregnant female using an interlabial pad. An interlabial pad(e.g., as included with the PADKIT® available from QuantRx, Corp., anddescribed herein is used to collect a biological sample from thepregnant subject, for example, as described in Examples 1 and 2 above.The biological sample is collected from the pad by centrifugation toform a pellet, which includes cells from the biological sample, as wellas other material. Cell pellets obtained from the interlabial padcontain both maternal cells and various types of fetal cells. Forexample, the fetal cells are shed from the placenta into the vaginalfluid and are captured by the interlabial pad. The fetal cells(including pluripotent human embryo stem cells) are separated from thematernal cells to facilitate study and use of the fetal cells, includinganalysis of the genome of the fetus. Once obtained, the pellet is storedat 4° C. until further processing.

A confirmation step can optionally be performed, to confirm that fetalcells are present in the cell pellet obtained from the interlabial pad.Fetal cells can be detected by determining if protein markers for fetalcells, such as alpha-fetoprotein (AFP), H-19 protein, yes associatedprotein (YAP65), and osteopontin, are present in a sample of the pelletobtained from the interlabial pad. Alternatively, fetal cells can bedetected by determining if embryonic stem cells are present in a presentin a sample of the pellet obtained from the interlabial pad, for exampleby determining if markers for embryonic stem cells (such as ssEA4,TRA-1-60, and TRA-1-81 proteins) are present on cells in the sample ofthe pellet obtained from the interlabial pad, for example by processingthe sample with an embryonic stem cell (ESC)/induced pluripotent stemcell (iPSC) characterization kit (e.g., as available from AppliedStemCell, Inc., Cat. No. ASK-3006) for human ESCs according to themanufacturer's directions. Antibodies for use to purify fetal cells fromthe pellet obtained from the interlabial can be selected based on thedegree and intensity of staining of fetal cells in the confirmationstep.

Fetal cells in the pellet obtained from the interlabial pad areisolated, e.g., from maternal cells in the pellet. Fetal cells can bepositively, and/or maternal cells can be negatively, selected using avariety of techniques well known in the art, including cell sorting, forexample by fluorescence activated cells sorting (FACS) and/or affinitypurification (such as using an affinity reagent bound to a solid phaseparticle which can be isolated on the basis of the properties of thesolid phase particles for example beads (e.g., colored latex beads ormagnetic particles). For example, the fetal cells can be separated frommaternal cells based on positive selection by FACS or affinitypurification using a fetal marker, such as expression ofalpha-fetoprotein (AFP), H-19 protein, yes associated protein (YAP65),or osteopontin protein. Further, fetal stem cells (such as ESCs) can beseparated from maternal cells based on positive selection by FACS oraffinity purification using a fetal ESC marker, such as expression ofssEA4, TRA-1-60, or TRA-1-81 protein

The pellet obtained from the interlabial pad is washed (e.g., threetimes) with appropriate buffer, such as PBS. The washed cells areincubated with an antibody specific for a fetal cell specific proteinmarker (such as AFP). The antibody is directly labeled with afluorescent marker (such as fluoroscein) or with a magnetic bead. Thelabeled cells are washed three times to remove non-specifically boundantibody.

In some examples, incubation of the antibody with the cells obtainedfrom the interlabial pad causes the cells to clump together. In thisevent, the clumped cells (including mostly cells bound by the antibody,that is, fetal cells) can be separated from non-clumped cells (includingmostly cells not bound by the antibody, that is, maternal cells) bylow-speed centrifugation. This additional purification step is typicallyperformed prior to separation of cells based on FACS or affinitypurification. The labeled (fetal) cells are sorted from non-labeled(maternal) cells using FACS (if the antibody is labeled with afluorescent marker) or magnetic separation (if the antibody is labeledwith a magnetic bead). The sorting step can be repeated multiple times(such as three times) to increase purity of the isolated fetal cells.The sorted fetal cells can be processed for further analysis (such asdetermination of the fetal genotype), and/or expanded in tissue culturefor future use.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described embodiments. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. A method of obtaining fetal cells, comprising placing an absorbentmedium in an interlabial or intravaginal space or adjacent to theperineum at the vaginal opening of a pregnant female with an intactamniotic sac, collecting vaginal fluid comprising cells in the absorbentmedium while the absorbent medium is in the interlabial or intravaginalspace or adjacent to the perineum at the vaginal opening; removing theabsorbent medium; extracting the vaginal fluid from the absorbentmedium; and isolating cells from the vaginal fluid extracted from theabsorbent medium, thereby obtaining the fetal cells.
 2. The method ofclaim 1, wherein the absorbent medium is in the form of a panty liner,an interlabial pad or a sanitary napkin.
 3. The method of claim 1,wherein the absorbent medium is placed in the interlabial space.
 4. Themethod of claim 3, wherein the absorbent medium is at least partially,or substantially entirely, placed in the interlabial space for about 2to about 6 hours.
 5. The method of claim 2, wherein the interlabial padincludes a major portion and a minor portion, and wherein the absorbentmedium is removed by gripping the minor portion and removing theinterlabial pad.
 6. The method of claim 1, wherein the absorbent mediumcomprises a container having a plurality of fluid receiving aperturestherein encompassing the absorbent medium, and wherein the absorbentmedium is configured for intravaginal placement in the pregnant female.7. The method of claim 1, wherein the absorbent medium comprises aninner core and an outer covering, the outer covering having a visiblematrix of pores of sufficient size to allow cells to enter the pores,and wherein the absorbent medium is configured for intravaginalplacement in the pregnant female.
 8. The method of claim 1, wherein theabsorbent medium comprises rayon, cellulose, cotton, other naturalfibers, or synthetic materials
 9. The method of claim 1, wherein thefetal cells are fetal stem cells.
 10. The method of claim 1, wherein thefetal cells are somatic cells, embroyic stem cells, fetal stem cells ortrophoblast cells.
 11. The method of claim 1, wherein isolating cellsfrom the absorbent medium comprises: releasing cells from the absorbentmedium with a physiological buffer; collecting the physiological buffer;centrifuging the physiological buffer to form a pellet of cellularmaterial; and suspending the cellular material in a physiologicalbuffer, thereby isolating the fetal cells.
 12. The method of claim 12,wherein releasing cells from the absorbent medium comprises the use ofvibration or soniciation.
 13. The method of claim 1, wherein isolatingcells from the vaginal fluid comprises separating the fetal cells frommaternal cells in the vaginal fluid.
 14. The method of claim 13, whereinseparating fetal cells comprises the use of an agent that specificallybinds the fetal cells.
 15. The method of claim 14, wherein the agent isan antibody.
 16. The method of claim 15, wherein the antibodyspecifically binds a major histocompatibility molecule.
 17. The methodof claim 1, further comprising analyzing the genome and/or epigenome ofat least one fetal cell at a locus of interest.
 18. The method of claim17, wherein analyzing the genome comprises determining the presence orabsence of a Y chromosome.
 19. The method of claim 1, wherein analyzingthe genome comprises the use of polymerase chain reaction.
 20. Themethod of claim 19, wherein analyzing the epigenome comprisesdetermining a degree of methylation or histone modification of anepigenomic locus.
 21. The method of claim 1, further comprisingculturing at least one fetal cell in a tissue culture medium.
 22. Themethod of claim 21, wherein the fetal cell is a stem cell.
 23. Themethod of claim 22, wherein the stem cell is a totipotent cell, apluripotent cell or a multipotent cell.
 24. The method of claim 22,further comprising propagating the stem cell in vitro.
 25. The method ofclaim 24, further comprising differentiating the stem cell in vitro. 26.A method of fetal diagnosis, comprising placing an absorbent medium inan interlabial or intravaginal space or adjacent to a perineum at avaginal opening of a pregnant female, collecting vaginal fluid in theabsorbent medium while the absorbent medium is in the interlabial orintravaginal space or adjacent to the perineum at the vaginal opening;removing the absorbent medium from the pregnant female; and subjectingcells in the absorbent medium to a fetal diagnostic test, therebydiagnosing the fetus.
 27. The method of claim 26, further comprising:isolating at least one fetal cell from the absorbent medium; andanalyzing genetic material isolated from the at least one fetal cell todetermine the presence or absence of a genomic or epigeneticcharacteristic associated with a biological outcome.
 28. The method ofclaim 26, wherein analyzing the genetic material comprises determiningthe presence or absence of the Y chromosome, wherein the presence of theY chromosome identifies the fetus as a male.
 29. The method of claim 26,wherein the pregnant female has an intact amniotic sac.
 30. The methodof claim 26, wherein subjecting cells in the absorbent medium to thefetal diagnostic test comprises a test performed by another party.
 31. Amethod of determining the sex of a fetus, comprising placing anabsorbent medium in an interlabial or intravaginal space or adjacent toa perineum at a vaginal opening of a pregnant female; collecting vaginalfluid in the absorbent medium while the absorbent medium is in theinterlabial or intravaginal space or adjacent to the perineum at thevaginal opening; removing the absorbent medium from the pregnant female;isolating at least one fetal cell or fetal genetic material from theabsorbent medium; and analyzing the genetic material or genetic materialisolated from the at least one fetal cell to determine the presence orabsence of a Y chromosome, wherein the presence of the Y chromosomeidentifies the fetus as a male and the absence of the Y chromosomeidentifies the fetus as a female; thereby determining the sex of thefetus.
 32. The method of claim 31, wherein the pregnant female has anintact amniotic sac.